Elevated fat levels within tissues and reduced capacity/inefficiencies in long-chain fatty acid (LCFA) oxidative catabolism are highly correlated with muscle, liver, adipose, and whole-body insulin resistance. Specific metabolites whose intra- or extra-cellular concentrations shift in response to changes in capacity or efficiency of mitochondrial LCFA combustion may act as bioactive molecules that impact insulin signaling. Our parent project is leveraging multiple metabolomics analysis platforms to discover new metabolites that correlate with muscle mitochondrial FA combustion and T2DM generally, and one interesting outcome to date has been the identification of medium-chain fatty acylcarnitines (MCFA-carnitines [C6-C14-carns], reflective of incomplete LCFA [unreadable]-oxidation) as entities increased in T2DM plasma. Proof-of-principle studies demonstrated activation by MCFA-carnitines of NF?B-related pro-inflammatory pathways in a murine macrophage cell line. Might the mechanism underlying the pro-inflammatory effect of MCFA-carnitines, acting locally or systemically, be via activation of upstream Toll-like receptors (TLRs), previously shown to be triggered by saturated FAs including C12:0 and C14:0 but not previously linked to acylcarnitines? Could such a mechanism underlie part of the insulin resistance associated with dysfunctional FA metabolism, as seen in many pre-diabetics and T2DM patients? Our novel preliminary results point to specific activation of the upstream cell-surface receptor Toll-like receptor 2 (TLR2) by MCFA-carnitines, and we anticipate that this in turn elicits insulin resistance phenotypes in target cells. If true, this would be a major step forward in understanding the etiology of T2DM, and in identifying the specific links between dysfunctional fatty acid metabolism and insulin resistance. The proposed project, which complements our parent project, has two primary Aims that will definitively answer if acylcarnitines evoke inflammation via TLR2 (and specifically, TLR2-TLR1 dimerization), and whether acylcarnitines can attenuate insulin action in vivo and in vitro through TLR2-dependent mechanisms. Specific Aim 1--Confirm that MCFA-Carnitines Specifically Activate TLR2-TLR1 Leading to Impaired Insulin Signaling. Studies to date using murine cell systems and murine TLR gene constructs suggest that C12-carn treatment increases NF?B-based pro-inflammatory gene expression through activation of TLR2-TLR1 heterodimerization but not other TLR systems. This aim will confirm and extend those results by: (a) evaluating if knockdown of TLR2 or its immediate downstream adaptor MyD88 reduces or abolishes the activation of inflammatory pathways by MCFA-carnitine, (b) testing if a TLR2 dominant-negative mutant (but not other TLR mutants) reduces the effects of MCFA-carnitine, (c) determining whether MCFA-carnitine induces TLR2-TLR1 heterodimerization, and (d) determining if MCFA-acylcarnitine can impair insulin signaling through TLR2-mediated pathways. Specific Aim 2--Determine if MCFA-Carnitine Administration Activates Pro-Inflammatory Pathways and Induces Insulin Resistance in vivo, Mediated Through TLR2-Dependent Mechanisms. No studies have examined effects of acylcarnitines on inflammation and insulin action in vivo. We predict that the inflammatory phenotype observed in cultured cells in response to C12-carn (Aim 1 &Preliminary Results) will be recapitulated in vivo, which would support our working hypothesis that local and/or systemic accumulation of acylcarnitines in T2DM exacerbates chronic inflammation and hence plays a role in the insulin resistance phenotype. These proof-of-concept studies will: (a) examine if acute i.v. injection of C12-carn in mice triggers TLR2/NF?B-dependent gene pro-inflammatory pathways in target tissues (muscle, liver, fat, blood leukocytes), and will determine if these outcomes are abolished or dampened in TLR2-KO mice, and (b) test if longer-term exposure to high systemic C12-carn increases tissue inflammatory markers and whole-body insulin resistance, with effects abolished or reduced in TLR2-KO mice. PUBLIC HEALTH RELEVANCE: A reduced ability of the pancreatic hormone insulin to trigger tissue uptake of blood sugar is an early event in the course of development of type 2 diabetes mellitus (T2DM). Relatively poor fat combustion by fasting muscle is often correlated with insulin resistance, even in the pre-diabetic state, and altered fat metabolism also appears to diminish insulin action in liver, fat tissue, and the insulin-producing cells of the pancreas. Thus, the overarching aim of our research is to identify clinically-relevant metabolite biomarkers of dysfunctional fat metabolism, and to understand if at least some of these factors not only mark disease susceptibility but also participate in diabetes causation or exacerbation. Specific to this project is the goal to follow-up on initial results indicating that naturally-occurring acylcarnitine metabolites (fatty acids of differing chain-lengths, bound to an amino-acid-like molecule), which are elevated in the T2DM blood as byproducts of inefficient fat metabolism, activate pro-inflammatory/insulin-resistance pathways through the specific cell-surface receptor Toll-like receptor 2 (TLR2). If true, this will have a profound influence on our understanding of diabetes pathophysiology and may provide new targets for preventive and therapeutic modalities.